1. Bridge Class Routing vectors to Genetic Engineering Introduction to Vectors

2. 1.Identification of genomic sequences Reverse/Forward genetics 2.Isolation of genomic sequences PCR, RT-PCR, genomic/cDNA libraries 3.Verification of genomic sequences Sequencing technologies 4.Manipulation of genes and genomic sequences Cutting, pasting, changing (Site directed mutagenesis) Tools: Enzymes 5.Assorting combinations from unrelated species Tools: Enzymes 6.Cloning of rDNA Making use of vectors and strains 7.Expressing heterologous biomaterial (proteins of therapeutic or economic value in microorganism to provide proof of concept, pilot scale, industrial level Defining the purview of genetic engineering?

3. Considerations prior to cloning (Plasmid Vectors) Source of the genomic fragment PCR amplified Taq polymerase Pfu polymerase Choice w.r.t Ligation strategies Released thru Restriction (frm another vector) TA Cloning TOPO-TA Cloning Blunt end Cloning Cohesive end Cloning S1 nuclease Forced ‘A’ tailing Linearaisation of vector with compatible RE CIAP Treatment of vector Linkers and adaptors Homopolymeric tailing of vector and insert

4. Standard methodology of cloning

5. Pipeline downstream of Ligation Ligation mix (mixture of recombinant and non- recombinant plasmids) Transformation Heat shock method Electro-transformation A competent E. coli strain Preparation of Host: Competent cells (Chemi compis or electro-compis) CaCl 2 method (Mandal and Higa, 1970) RbCl 2 Method (Hanahan, 1983) Other protocols (eg For electro-competent, glycerol)

6. E.coli Transformation: Introduction of DNA into host cells Classical definition: Natural uptake of naked ds DNA by bacterial cells. Fred Griffiths (1928) Streptococcus pneumoniae (a.k.a. Pneumonococcus or Diplococcus) Avery, McCarty and MacLeod (1944) proved that DNA is the transforming principle. Competence Only competent cells can take up DNA from external mileu Competent state induced in response to nutritional shut down Not all bacteria are competent, all the time (E. coli not naturally competent) Natural competence Artificially induced

7. E. coli K12: laboratory host for rDNA Ensuring Safety in Molecular Biology laboratory/human health/ environment Outcome of Asilomar Conference (Center on California’s Monterey Peninsula 1976) Scientific fraternity realizing the importance of working with “safe” strains of bacteria, viruses and vectors Scientific fraternity realizing the importance of working with “safe” strains of bacteria, viruses and vectors Containment to be made a part of expt design, level of containment proportional to risk involved/biohazard (Risk assessment and analysis) Containment to be made a part of expt design, level of containment proportional to risk involved/biohazard (Risk assessment and analysis) NIH formed the Recombinant DNA Advisory Committee (RAC) NIH formed the Recombinant DNA Advisory Committee (RAC) It took 1 year (1976) before the first “safe” (EK2 category) line of E. coli was released It took 1 year (1976) before the first “safe” (EK2 category) line of E. coli was released That year, RAC released a set of guidelines requiring the use of safe bacteria That year, RAC released a set of guidelines requiring the use of safe bacteria